Carcinogenic nitrosamines are activated via alpha-hydroxylation to reactive alkylating agents. In the case of asymmetric nitrosamines there are two potential activation pathways. Interactions between the two pathways have not been studied. This question will be addressed using two asymmetric nitrosamines 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) and nitrosobenzylmethylamine (NBzMA). Both compounds are activated to either a methylating agent or a bulky alkylating agent. Experimental data support a carcinogenic mechanism for these two compounds in which the formation of O6-methylguanine (O6-mG) is important for the initiation of tumors in target tissues. The bulky DNA adducts formed during the activation of these two carcinogens are capable of inactivating the O6-mG repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). The hypothesis tested in this proposal is that the bulky adducts formed by these compounds act as co-carcinogens for the methylation pathway by increasing the initiating activity of O6-mG through AGT inactivation. The mechanism by which these bulky adducts inactivate AGT will be investigated. In addition, the ability of these adducts to compete with O6-mG for reaction with AGT will be examined. Studies have been designed to investigate the ability of these pathways to inactivate AGT in vivo and to determine the contribution of this reaction to O6-mG persistence in target tissues. The ability to increase O6-mG persistence will be related to tumor incidence using the A/J mouse lung tumor model. Evidence for these co-carcinogenic pathways will be sought in other susceptible species.